Using molecular modeling and NMR spectroscopy, the three-dimensional structures of wild type and mutant homeodomain containing proteins in the NK-2 class, the human CSX/NKX-2.5, and NKX-3.1 proteins, and the unique HOP homeodomain, have been investigated in the free state, bound to DNA, and in ternary complexes with associated transcription factors. The most recent results focus on the structural mechanisms of DNA specificity and enhancer complex assembly, including interaction of human Nkx3.1, CSX/NKX-2.5 and HOP with serum response factor (SRF). NMR of these homeoproteins have revealed novel interaction motifs and suppression mechanisms playing crucial roles in cardiogenesis, which have subsequently been confirmed by functional assays with mutations in the regions identified by NMR. Investigation of the human homeodomain T41M mutant of Nkx3.1, an oncogene found to co-segregate with prostate cancer risk, showed that the homeodomain was thermally destabilized, though its structure and DNA binding were otherwise unaltered. NMR and calorimetry studies comparing the human CSX/NKX-2.5 homeodomain and Drosophila NK-2 proteins have resolved long-standing questions on the role of protein stability and temperature dependence on DNA binding free energy.